Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Avda. de las Ciencias, s/n, 10003 Cáceres, Spain.
Food Hygiene and Safety, Meat and Meat Products Research Institute, Faculty of Veterinary Science, University of Extremadura, Avda. de las Ciencias, s/n, 10003 Cáceres, Spain.
Int J Food Microbiol. 2018 Jun 20;275:17-23. doi: 10.1016/j.ijfoodmicro.2018.03.020. Epub 2018 Mar 24.
The cell wall integrity (CWI) pathway is activated in response to cell wall stresses due to different food-related environments. Rho1 is one of the main regulators within such pathway. The objective of this work was to design an easy-to-use RT-qPCR technique for the evaluation of the Rho1 gene expression useful to measure responses to the presence of cell wall stressors such as the antifungal protein PgAFP. Two primer pairs were designed from published conserved regions. Their specificity initially was determined by in silico analysis for several fungal species. After optimising the qPCR, the primer pair Rho1-F1/R2 was selected due to the lowest Cq values obtained and its specificity. The qPCR method showed efficiencies between 97.5% and 100.5%. Applicability of the designed qPCR method was evaluated in the presence of the stressor PgAFP. The PgAFP-resistant Penicillium polonicum and the PgAFP-sensitive Aspergillus flavus showed Rho1 gene over- and under- expression, respectively, indicating that the CWI pathway is activated in the former species but not activated in the latter one in response to the stress caused by PgAFP. This novel qPCR methodology able to detect changes in CWI-related gene expression in filamentous fungi will be useful in future studies to evaluate physiological mould responses to different food environmental challenges.
细胞壁完整性 (CWI) 途径会在受到与食物相关的不同环境中的细胞壁压力时被激活。Rho1 是该途径中的主要调节剂之一。本工作的目的是设计一种易于使用的 RT-qPCR 技术，用于评估 Rho1 基因表达，以测量对细胞壁应激物（如抗真菌蛋白 PgAFP）存在的反应。根据已发表的保守区域设计了两对引物。通过对几种真菌物种的计算机分析初步确定了它们的特异性。在优化 qPCR 后，由于获得的最低 Cq 值及其特异性，选择了引物对 Rho1-F1/R2。qPCR 方法的效率在 97.5%至 100.5%之间。在存在应激物 PgAFP 的情况下，评估了设计的 qPCR 方法的适用性。对 PgAFP 具有抗性的波兰青霉和对 PgAFP 敏感的黄曲霉分别表现出 Rho1 基因的过表达和低表达，表明 CWI 途径在前者中被激活，但在后者中未被激活，以应对 PgAFP 引起的应激。这种新型 qPCR 方法能够检测丝状真菌中与 CWI 相关的基因表达变化，将有助于未来评估不同食物环境挑战下霉菌的生理反应。
